Ethical and Social Considerations of Mitochondrial Replacement Therapy (MRT)

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Ethical and Social Considerations of
Mitochondrial Replacement Therapy (MRT)

• Committee on Ethical and Social Policy
  Considerations of Novel Techniques for Prevention
  of Maternal Transmission of Mitochondrial DNA
  Diseases
• Public Workshop
• March 31-April 1, 2015

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Ethical and Social Considerations of MRT

• Kinship
• Value of / preference for genetically-related
  children
• Existence of alternative approaches to parenting
• Influence of reproductive autonomy in the US
• DNA from three individuals
• Social considerations
• Identity
• Parenthood
• Ancestry

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Ethical and Social Considerations of MRT

• First-in-human research for the purpose of
  creating children
• Informed consent for an unborn child consent for
  future generations
• Children and women are bearers of risk
• Disease prevention vs reproductive
  opportunity (non-identity)
• Curing a child otherwise born with mito disease
  or creating a new person?
• Allowable risk vs perceived benefit?

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Ethical and Social Considerations of MRT

• Risk to women (potential mothers and egg donors)
• Ovarian hyperstimulation syndrome (OHSS)
• Risk to embryo
• Epigenetic modification
• Reagents used (e.g. dissolving agent in MST)
• Risk to offspring
• Creation of disease through alleviation of
  another
• Birth, developmental, long-term defects
• Haplotype incompatibility (sterility?)
• mtDNA carryover ? heteroplasmy
• Risk to future generations
• mtDNA carryover ? mtDNA bottleneck ? ?
  heteroplasmy in oocytes

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Ethical and Social Considerations of MRT

• Moral status of oocytes and embryos
• Manipulation and/or destruction of oocytes vs
  embryos
• (MST or PB1T ) vs (PNT or PB2T)
• Considerations of relative safety of each
  technique

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Ethical and Social Considerations of MRT

• Fairness, equity, and access
• Fee-for-service assisted reproductive
  technologies
• Increased demand for egg donors payment
• Availability of alternative options (adoption or
  egg donation)
• Desire for genetically related children?

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Ethical and Social Considerations of MRT

• Downstream applications
• Disease threshold for inclusion criteria?
• Creation of an obligation for at-risk families?
• Acceptable range of potential genetic
  modifications introduced?
• Treatment vs enhancement
• Impact on acceptance of nDNA germline
  modification?

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Ethical and Social Considerations of MRT

• Germline modification
• Working definition human inheritable genetic
  modification (FDA)
• nDNA vs mtDNA a distinction for germline
  modification?
• Distinction between replacement and editing of
  DNA?
• Controls therapeutic vs enhancement

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Maternal Spindle Transfer (MST)

1. The spindle of chromosomes is removed from the
   donor egg and discarded.
2. The spindle of chromosomes is removed from the
   intending mothers egg and transferred to the
   enucleated donor egg the intending mothers
   egg is discarded.
3. The reconstructed oocyte contains the intending
   mothers nuclear DNA and donors mitochondrial
   DNA.
4. The egg is then fertilized with the intending
   fathers sperm.
5. The embryo develops in vitro and is transferred
   to the womb of the woman who will carry the
   child.

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(Nuffield Council on Bioethics, 2012)
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Maternal Spindle Transfer (MST)

• Potential risks
• mtDNA carryover PBT lt MST lt PNT (estimated lt1)
• Technicality of procedure
• Spindle-chromosome complex sensitive to
  manipulation higher risk of chromosomal
  abnormalities than in PNT
• Visualization of spindle
• Operator dependent
• Reagents treatment of oocytes with cytoskeletal
  inhibitors for karyoplast removal Sendai virus
  for fusion
• Ethical considerations
• Manipulation and destruction of oocytes
• nb Embryos deemed not suitable for transfer may
  be discarded.

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Maternal Spindle Transfer (MST)
State of the science
Study Model Endpoint Methods Results Summary
Tachibana et al. (2009) Rhesus macaques (Macaca mulatta) 'genetically distant sub-populations' Developmental potential F1 health, mtDNA carryover 15 ST embryos transferred into 9 ? 6 with 1-2 blastocysts, 3 with 2 cleavage stage (4-8 cell) embryos Four healthy offspring born following blastocyst transfer (one set of twins, two singletons) 3 carryover of mtDNA The ST strategy will probably result in least amount of mtDNA carryover, as compared to other techniques ST could present a reliable approach to prevention of transmission of mtDNA mutations
Tachibana et al. (2013) (2009 follow-up) Rhesus macaques Overall health Post-natal development Routine blood and bodyweight measurements (birth 3 years) Normal development No change in mtDNA carryover and heteroplasmy in blood and skin samples Oocyte manipulation and mtDNA replacement procedures are compatible with normal development Nuclear mtDNA interactions conserved within species
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Maternal Spindle Transfer (MST)
State of the science, cont.
Study Model Endpoint(s) Methods Results Summary
Tachibana et al. (2013) Human oocytes Developmental potential mtDNA carryover 106 donated oocytes 65 ST, 33 control Reciprocal ST followed by ICSI Significant portion of ST oocytes (52) showed abnormal fertilization remaining normally fertilized ST zygotes had comparable level of blastocyst development (62) lt1/ND carryover of mtDNA in ST embryos Human oocytes are more sensitive to spindle manipulations than macaques Compared to ST in macaque oocytes, ST in human oocytes resulted in a significant level of abnormal fertilization
Paull et al. (2013) Human oocytes Preimplantation development mtDNA carryover 62 donated oocytes partheno-genetically activated Efficient development to blastocyst stage (37 vs 32 control) mtDNA carryover 0.5, decreased to ND in blastocysts and eSC Depolimerization prevents premature oocyte activation MST did not reduce developmental efficiency to blastocyst stage and resulted in carryover of lt1, which decreased to ND Spontaneous activation of oocytes can be avoided by cooling the spindle complex
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Maternal Spindle Transfer (MST)
State of the science, cont.
Study Model Endpoint Methods Results Summary
Lee et al. (2012) Rhesus macaque oocytes Developmental potential of ST embryos Level of heteroplasmy in somatic tissues of preterm fetus (F1) and oocytes (F2), 135d post-embryo transfer 102 ST oocytes generated Two singleton pregnancies generated using preselected ? embryos 63 of ST developed to blastocysts after fertilization mtDNA carryover lt0.5/ ND in somatic tissues of F1 11/12 oocytes in each fetus (F2 generation) displayed ND levels of mtDNA heteroplasmy one oocyte from each fetus contained substantial mtDNA carryover (16.2 and 14.1) Confirms that MST results in low level of mtDNA carryover Supports the observation that different mtDNA transmission mechanisms may exist for somatic and germline lineages
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Pronuclear Transfer (PNT)

1. The intending mothers egg is fertilized by the
   intending fathers sperm.
2. The donor egg is also fertilized by the intending
   fathers sperm.
3. The pronuclei are removed from the single-celled
   zygote of the donor egg and discarded.
4. The pronuclei are removed from the intending
   mothers fertilized egg and transferred to the
   enucleated fertilized donor egg. The enucleated
   fertilized egg of the intending mother is
   discarded.
5. The reconstructed embryo contains pronuclear DNA
   from the intending parents and healthy
   mitochondria from the donor.
6. The embryo develops in vitro and is transferred
   to the womb of the woman who will carry the
   child.

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(Nuffield Council on Bioethics, 2012)
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Pronuclear Transfer (PNT)

• Potential risks
• mtDNA carryover PBT lt MST lt PNT (estimated lt2)
• Technicality of procedure
• Easier visualization than MST (enclosed in
  karyoplast)
• Need to ensure inclusion of centrioles and other
  spindle assembly components
• Operator dependent
• Reagents treatment of zygotes with cytoskeletal
  inhibitors for karyoplast removal Sendai virus
  for fusion
• Ethical considerations
• Manipulation and destruction of fertilized eggs

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Pronuclear Transfer (PNT)
State of the science
Study Model Endpoints Method Results Summary
Sato et al. (2005) Mito-mice (?mtDNA Mus musculus domesticus) Wild-type mice Mus spretus Rescue from disease phenotype mtDNA carryover 39 mito-mouse zygotes transferred into pseudo-pregnant females 34 control (mito-mouse, no PNT) 11 mice born following PNT (9 control) F0 progeny rescued from disease phenotypes Average carryover 11 at weaning, increased to 33 gt300d estimated to be 43 at day 800 PNT is restricted to patients with mitochondrial diseases wherein pathogenic mtDNAs inherited maternally and do not possess significant replication advantages over wild-type mtDNA
Craven et al. (2009) Human zygotes (abnormally fertilized unipronuclear/ tripronuclear) Developmental potential mtDNA carryover Pronuclei (2) transferred to enucleated recipient zygote monitored 6-8 days in vitro 22 developed past 8-cell stage, 8.3 to blastocyst stage (50 of unmanipulated control) mtDNA carryover lt2/ND PNT has the potential to prevent mtDNA disease transmission and results in very low mtDNA carryover
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Pronuclear Transfer (PNT)
State of the science
Study Model Endpoints Method Results Summary
Turnbull group, unpublished Human zygotes (normally fertilized) Developmental potential mtDNA carryover Unavailable High rates of development to blastocyst stage mtDNA carryover lt2/ND Modifications to experimental protocol resulted in increased development to blastocyst stage
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Oogenesis Formation of Polar Bodies

• The primordial germ cell (oogonium) undergoes
  mitosis in the fetus at birth, the primary
  oocyte arrests in prophase of meiosis I (prophase
  I).
• Beginning at puberty, once per month, a primary
  oocyte completes meiosis I and begins meiosis II,
  before arresting at metaphase II. At this time
  the first polar body is produced. The resultant
  secondary oocyte and polar body are haploid.
• The secondary oocyte is ovulated. If fertilized
  by a sperm, the secondary oocyte completes
  meiosis II and the second polar body (haploid) is
  formed.

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Polar Body 1 Transfer (PB1T)

• The chromosome spindle is removed from the donor
  egg and discarded.
• The 1st polar body is removed from the intending
  mothers egg and transferred to the enucleated
  donor egg the intending mothers egg is
  discarded.
• The reconstructed oocyte contains the intending
  mothers nuclear DNA and donors mitochondrial
  DNA.
• The reconstructed egg is fertilized with the
  intending fathers sperm.
• The embryo develops in vitro (PB2 extruded) and
  is transferred to the womb of the woman who will
  carry the child.

(Wolf et al., 2014)
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Polar Body 1 Transfer (PB1T)
State of the science
Study Model Endpoints Method Results Summary
Wang et al. (2011) Porcine Developmental potential Vitrified PB1 T 88.6 normal recombinant oocytes 9.3 cleaved 8-cell stage those that cleaved had normal morphology Frozen-thawed PB1s support oocyte fertilization and embryonic development
Wang et al. (2014) Mouse (Mus musculus) Developmental potential (in vitro in vivo) mtDNA carryover (F1 F2 generations) 25 PB1s 27 spindle-chromosome complexes transferred 14 PB1 and 18 ST embryos transferred to pseudopregnant ? mtDNA carryover tail tip/brain tissue and internal organs (F1) and toe tips (F2) PB1/ST 87.5/85.7 developed to blastocyst 42.8/44.4 live, healthy births ND/5.5 mtDNA carryover (tail tip/brain) ND/0-6.88 mtDNA carryover (internal organs) ND/7.1 mtDNA carryover (F2 generation) Proof for possibility of using MST in combination with PB1T to inc. chance of MRT success PB1T resulted in undetectable levels of heteroplasmic DNA in F1 and F2 generations
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Polar Body 1 Transfer (PB1T)
Potential risks mtDNA carryover PB1T lt PB2T lt
MST lt PNT Technicality of procedure potentially
easier to obtain polar bodies, as they are
already enclosed in their own cell membrane can
be removed with only micropipette Ethical
considerations Manipulation and destruction of
oocytes nb embryos deemed not suitable for
transplant may be discarded.
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Polar Body 2 Transfer (PB2T)

• The intending mothers egg is fertilized by the
  intending fathers sperm. (not shown)
• The donor egg is fertilized by the intending
  fathers sperm. (not shown)
• The maternal pronuclei from the donor zygote is
  removed and discarded, leaving a half-enucleated
  egg.
• The 2nd polar body from the intending mothers
  zygote is transferred to the half-enucleated
  donor egg, which contains the paternal pronuclei
  and donor mtDNA.
• The embryo develops in vitro and is transferred
  to the womb of the woman who will carry the child.

(Wolf et al., 2014)
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Polar Body 2 Transfer (PB2T)

• Potential risks
• mtDNA carryover PB1T lt PB2T lt MST lt PNT
• Technicality of procedure
• Identification of female pronuclei
• Potentially easier to obtain polar bodies, as
  they are already enclosed in their own cell
  membrane can be removed with only micropipette
• Ethical considerations
• Manipulation and destruction of fertilized eggs
• nb embryos deemed not suitable for transplant
  may be discarded.

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Polar Body 2 Transfer (PB2T)
State of the science
Study Model Endpoints Method Results Summary
Wakayama et al. (1997) Mouse (Mus musculus) Integrity of PB genomes Developmental potential Effect of timing PB2T with PB2 from same or different oocyte Transfer of 30 compacted murulae or blastocysts to six pseudopregnant ? Reconstructed embryos had well-developed pronuclei Developmental rate decreased as time of PB2 transfer after fertilization increased (70 when recently fertilized) 18 live, healthy births The timing of transfer is important to success PB2T supports full term embryo development and therefore could be used as an alternative source of female chromosomes
Wang et al. (2014) Mouse (Mus musculus) Developmental potential mtDNA carryover (F1 F2 generations) 30 PB2s 38 pronuclei transferred 15 PB2 and 13 PNT embryos transferred to pseudopregnant ? mtDNA carryover examined in tail tip/brain tissue and internal organs (F1) and toe tips (F2) PB2/PNT 55.5/81.3 developed to blastocyst 40/53.8 live, healthy births 1.7/23.7 mtDNA carryover (tail tip/brain) ND-3.62/5.5-39.8 mtDNA carryover (internal organs) 2.9/22.1 mtDNA carryover (F2 toe tip) PB2T results in very low level mtDNA carryover PB1T, PB2T and ST could be readily used to exchange mtDNA